The Interactive Fly
Zygotically transcribed genes

DNA Replication

Timing of DNA Replication

DNA Repair


DNA polymerases and subunits

Origin recognition complex

Mini-chromosome maintenance family

DNA replication factor A complex - a single stranded DNA-binding protein complex

Other proteins

How does DNA replication take place?

Why should a developmental biologist be interested in DNA replication? There are at least three reasons. (1) For a given origin of replication, there is a link between gene expression and timing of DNA replication, and understanding the basis of this link is important. (2) The mechanism of gene replication is by necessity involved with the restructuring of chromatin and the regulatory implications of that event. (3) There are fail-safe mechanisms to ensure that each origin of replication fires only once per cell cycle, and these mechanisms involve an interaction of cyclins with licensing factor, a chromatin component. Thus, in the future there will be a developing understanding of the relationship between cell cycle, DNA replication, chromatin components, and changes in gene expression.

The first signal for initiation of replication involves replication licensing factor (RLF), which 'licenses' replication origins by putting them into an initiation-competent state. The second signal, S-phase promoting factor, induces licensed origins to initiate, and in doing so removes the license. RLF of Xenopus can be separated into two essential components, RLF-M and RLF-B, both of which are required for licensing. RLF-M, a fraction containing members of the minichromosome maintenance family, associates with chromatin prior to replication but is removed during replication. Drosophila MCM2 and MCM4 homologs have been identified (See disc proliferation abnormal for information about both of these). RLF-M's reassociation with chromatin requires passage through mitosis. RLF-M requires RLF-B, an as yet uncharacterized fraction, for binding RLF-M to DNA. Apparently RLF binds to origins of replication, but the basis for this binding has not yet been characterized (Chong, 1996 and references).

The focus of all replication forks is the helicase, which catalyzes the transition from double- to single-stranded DNA. In eukaryotes, the identification of the enzyme that acts at chromosomal replication forks awaits further investigation, but the SV40 T antigen fulfills the helicase function in SV40 replication. The origin of replication is selected and identified in yeast by the origin recognition complex, of which one Drosophila homolog (Orc2) has been identified. Identification of other components of the ORC and unraveling the nature of DNA sequences at the origin are currently very active subjects of research.

The replication process is semidiscontinuous. Proteins comprising the replication machine act in concert to unwind the parental strands and carry out the simultaneous synthesis of the two progeny strands. Both progeny strands are synthesized in the 5' to 3' direction, but since parental DNA strands are antiparallel, two distinct mechanisms of DNA synthesis are required. One of the two progeny strands (the leading strand) is synthesized continuously in the direction of fork movement. The other (the lagging strand) is synthesized discontinuously in the direction opposite to fork movement. Discontinuous DNA synthesis on the lagging-strand templates involves the related synthesis of oligoribonucleotide primers, which are then elongated into short DNA chains (Okazaki fragments). Following their synthesis, Okazaki fragements are processed to remove the RNA primers and joined together to form an interrupted progeny strand (Brush, 1996).

As the replication fork advances, a helix-destabilizing protein is required to maintain the single-stranded DNA structure that serves as template for RNA priming and DNA synthesis. In eukaryotes, Replication protein A performs this function. This phosphoprotein consists of three subunits.

DNA synthesis is initiated by the bifunctional pol-alpha:primase complex, a heterotetrameric phosphoprotein. The primase activity resides in the smallest subunit and is tightly associated with the next largest, which is thought to tether the primase to the catalytic subunit. The remaining subunit has no known catalytic function, but it may contribute to recruitment of pol-alpha:primase to the replication fork. The main function of pol-alpha:primase is to serve as a priming enzyme. The primase catalyzes the synthesis of complementary oligoribonucleotides, which are then extended a short distance by the polymerase activity. The pol-alpha:primase serves exclusively to initiate DNA synthesis on the lagging strand, and dissociation from the DNA provides a primer terminus for assembly of the PCNA/pol-delta complex, which serves to extend the RNA/DNA primers originally synthesized by pol-alpha:primase.

The heterodimeric DNA polymerase-delta is involved in the elongation stage of DNA replication, acting both on the leading and lagging strands. Unlike pol-alpha:primase, the polymerase-delta does not act alone but requires the action of two auxiliary factors, the multisubunit replication factor C (RF-C) which binds to the primer terminus of the lagging strand immediately after RNA/DNA primer synthesis has been completed by pol-alpha:primase, allowing the subsequent assembly of a functional pol-delta complex. Once bound the primer-template junction, RF-C loads PCNA onto the DNA in an energy-dependent process. PCNA then functions as a processivity factor, acting as a clamp to prevent sliding of polymerase in the reverse direction. RNase H1 acts with another enzyme to remove the RNA primer used to initiate the lagging strand, while DNA ligase I joins the nacent DNA fragments to complete the synthesis of the lagging strand.

It is not clear how pol-alpha:primase synthesized DNA is proofread, as this enzyme has no exonuclease activity. the catalytic subunit of polymerase-delta contains a 3' to 5' exonuclease activity, functioning in proofreading, which allows high-fidelity DNA synthesis (Brush, 1996).

Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome

Duplication of eukaryotic genomes during S phase is coordinated in space and time. In order to identify zones of initiation and cell-type- as well as gender-specific plasticity of DNA replication, replication timing, histone acetylation, and transcription was profiled throughout the Drosophila genome. Two waves of replication initiation were identified with many distinct zones firing in early-S phase, along with multiple, less defined peaks at the end of S phase, suggesting that initiation becomes more promiscuous in late-S phase. A comparison of different cell types revealed widespread plasticity of replication timing on autosomes. Most occur in large regions, but only half coincide with local differences in transcription. In contrast to confined autosomal differences, a global shift in replication timing occurs throughout the single male X chromosome. Unlike in females, the dosage-compensated X chromosome replicates almost exclusively early. This difference occurs at sites that are not transcriptionally hyperactivated, but show increased acetylation of Lys 16 of histone H4 (H4K16ac). This suggests a transcription-independent, yet chromosome-wide process related to chromatin. Importantly, H4K16ac is also enriched at initiation zones as well as early replicating regions on autosomes during S phase. Together, this study reveals novel organizational principles of DNA replication of the Drosophila genome and suggests that H4K16ac is more closely correlated with replication timing than is transcription (Schwaiger, 2009).

The high resolution of these replication timing profiles allowed zones of replication initiation to be identified throughout S phase, that were confirmed in combination with measuring small nascent strand abundance. This revealed that sites of early initiation are rather distinct, which manifests in the timing profile as single peaks or a few peaks clustered together in early-S phase, followed by long stretches of the replication timing profile without changes in slope. Late initiation zones often reside in close proximity to other late initiation zones. The feature of distinct peaks of early initiation in Drosophila is very distinct from mammalian genomes (Hiratani, 2008), where many more sites of initiation of similar timing are clustered together, resulting in large regions up to several megabase pairs of early replication timing (Schwaiger, 2009).

Interestingly, the frequency of initiation appears discontinuous with high rates in early-S, a reduced frequency in mid-S, and again increased appearance of initiation sites in late-S phase. The high frequency and proximity of late-firing initiation zones suggest that late regions are replicated by many proximal late-firing origins of replication. This finding is particularly interesting in light of a recent report that suggested the absence of a checkpoint to control for the completion of DNA replication before mitosis (Torres-Rosell, 2007). This would in turn require a mechanism that mediates rapid replication of unreplicated regions in late-S phase, which could be achieved by a promiscuous activation of many proximal origins. Interestingly, replicative stress that reduces replication fork progression leads to a decrease in inter-origin distance through activation of normally dormant origins. It is conceivable that a similar situation is encountered in late replicating regions (Schwaiger, 2009).

Since the previously reported correlation between replication timing and transcription in Drosophila was not absolute, the percentage of the genome that replicates in a tissue-specific fashion remained to be tested quantitatively. For example, the general correlation could be driven by housekeeping genes that are active in most cells, resulting in a uniform replication timing program. This study showed that dynamic replication timing differs significantly between two Drosophila cell types, affecting at least 20% of autosomal DNA. It was also shown by two different methodologies that this plasticity of DNA replication coincides with transcription differences in only half of all cases (Schwaiger, 2009).

Early replication was shown previously to correlate with transcription levels over 180 kb, leading to the suggestion that replication timing integrates transcription over large regions. Consistent with this model, it was found that dynamic replication timing often occurs in large (~100 kb) regions encompassing many genes. Interestingly, genes with related function often cluster together in the Drosophila genome, and such clusters tend to be similarly 100 kb in size. In mammalian genomes, this clustering appears functionally related to chromatin structure, suggesting that widespread open chromatin at developmentally regulated multigene loci could lead to early replication or vice versa. This, in turn, might increase the potential of gene expression over large regions as in the case of genes important for wing disc development in Cl8 cells, where early replication could render the locus poised for activation (Schwaiger, 2009).

Localized differences in gene expression of a fraction of genes in a large region might also account for replication timing differences. Indeed, some, but not all, genes in differentially replicating regions are strongly differentially expressed between the two cell types. Thus, while gene expression could account for much of the observed changes on autosomes, a considerable fraction does not display transcriptional changes. It seems unlikely that the analysis missed such changes since noncoding transcription was measured as well as RNA polymerase abundance (Schwaiger, 2009).

The relation between replication timing and chromatin structure has been controversial. Transcription itself involves an opening of chromatin structure, and thus early replication could in many situations be downstream from transcriptional activation. However, previous work using injected plasmids suggested a role for early replication in mediating increased levels of histone acetylation. This led to a model in which replication timing mediates an open chromatin structure required for transcription. This suggestion is compatible with the genome-wide analysis, where a preferential location of H4K16ac was observed not only to active genes, but also to early replicating regions that are not transcribed. It is possible that early replication and elevated H4K16ac at inactive genes will result in a more open chromatin confirmation compared with late replicating inactive genes. This might render them more responsive to downstream activating cues, and thus replication timing could modulate the sensitivity to activators. This process could also function in maintenance of an active state through cell division. Importantly, however, this mechanism does not override the parallel process of transcription-coupled acetylation, as late replicating genes that are actively transcribed are still hyperacetylated (Schwaiger, 2009).

Interestingly, a strong abundance of H4K16ac was observed at sites of initiation during S phase. Several single-gene studies have suggested a positive function of histone acteylation for origin activity. Other reports, however, did not support this model. Recent maps of human replication initiation suggest that early origins are marked by H3K9/K14 acetylation (Lucas, 2007). However, no genome-wide correlation between active chromatin marks and early origin firing was observed in S. cerevisiae, where specific sequences function as origins of replication. This study identified a preferential localization of H4K16ac to initiation zones throughout the Drosophila genome compatible with a function of acetylation. In this study, focus was placed on acetylation of H4K16 because this residue has been functionally linked to higher-order chromatin compaction and chromatin opening on the dosage-compensated X in Drosophila (Schwaiger, 2009).

It has been proposed that origins of replication lie frequently between promoters of active genes, which would make transcription and replication fork progression co-oriented. Furthermore, transcription and replication are thought to be coordinated in the nucleus to be spatially and temporally separated. It thus seems plausible that the enrichment of H4K16ac in initiation zones reflects location between highly acetylated, active promoters. According to this model, proximity to active promoters would result in an open chromatin confirmation through increased H4K16ac, which in turn enhances origin firing (Schwaiger, 2009).

Importantly, however, enrichment for H4K16ac was observed at initiation zones that are not proximal to active genes, arguing against a simple process that is solely transcription-coupled. Open chromatin structure, reflected and potentially even mediated by H4K16ac, could make DNA more accessible for efficient initiation of DNA replication and thus provide a sequence-independent component that could contribute to origin localization and activity. While these are testable models, they do require a fine-mapping of actual origins at a resolution higher than the current detection of zones of initiation at the level of several kilobases (Schwaiger, 2009).

This analysis reveals the almost complete absence of late replication on the single X chromosome in male Drosophila cells. About 90% of female late replicating regions on the X replicate early in males, while autosomes show no advanced replication. Such chromosome-wide advance in replication timing has not been observed previously. In mammals, transcriptional inactivation of one of the female X chromosomes correlates with its late replication, reflecting the efficient silencing of this chromosome and increased chromatin compaction. In contrast, dosage compensation in flies involves the twofold up-regulation of genes already active in females and an open chromatin state mediated by H4K16ac. Interestingly, this study showed that advanced replication of the dosage-compensated X occurs mostly outside of transcriptionally activated regions and thus is unlikely to be accounted for by transcriptional changes. Importantly, the local increases in H4K16ac, which are detected throughout the male X chromosome, can be directly related to this loss of late replication. Reduction of the responsible Histone-Acetyltransferase Mof leads to a block in cell division, making it difficult to test this model. Notably, slightly delayed replication of the X chromosome was detected in the few cells that were in S phase in the knockdown population. While this is compatible with a model that Mof-mediated H4K16 acetylation advances replication of intergenic regions on the male X chromosome, the predominant effect on the cell cycle precluded further analysis (Schwaiger, 2009).

This suggests a transcription-independent, chromatin-dependent process, which leads to early replication chromosome-wide. While this likely reflects a different chromatin compaction, it is tempting to speculate that it also reflects a particular nuclear organization as the dosage-compensated X chromosome has been shown to associate directly with nuclear pores (Schwaiger, 2009).

Together these findings provide new principles of the replication timing program of the Drosophila genome and its dynamics relative to histone acetylation and transcription. The data further support a model in which open chromatin structure is a general feature of early replication timing and could potentially even advance replication of entire chromosomes (Schwaiger, 2009).

Genetic organization of interphase chromosome bands and interbands in Drosophila melanogaster

Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. This paper analyzed 32 cytology-mapped polytene chromosome interbands. Molecular locations of these interbands was estimated, their molecular and genetic organization was described and it was demonstrated that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential 'head-to-head' orientation of genes. They are enriched for 'broad' class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called 'grey' bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. A special algorithm was developed to computationally process protein localization data generated by the modENCODE project; it was shown that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date (Zhimulev, 2014. PubMed ID: 25072930).

DNA copy-number control through inhibition of replication fork progression

Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein Suppressor of Under-Replication (SuUR) through an unknown mechanism. This study demonstrates that SuUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. These results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression (Nordman, 2014: PubMed).

Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication

Among other targets, the protein lysine methyltransferase PR-Set7 (see Drosophila SET domain containing 7) induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. This study shows that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, it was further demonstrated that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase (see Drosophila MCM5) loading and replication activation at defined origins. Consistent with this, it was found that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes (Brustel, 2017).

Similarity in replication timing between polytene and diploid cells is associated with the organization of the Drosophila genome

Morphologically, polytene chromosomes of Drosophila melanogaster consist of compact 'black' bands (ruby bands, rb-bands) alternating with less compact 'grey' bands and interbands. This study developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, the replication timetable was obtained for all the bands mapped. The results allowed comparison of replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the Drosophila genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in Drosophila (Kolesnikova, 2018).

This work used the four-state chromatin model, previously published data on the chromatin localization of proteins, and in situ hybridization of annotated genes and identified the locations of all black bands of polytene chromosome 2R on a genome map (Kolesnikova, 2018).

The special feature of the four-state chromatin model is the generalization of data obtained from four cell lines. This generalization resulted in identification of two chromatin types -- aquamarine and ruby -- which show stable properties in all these cell lines. Starting the current mapping effort with identification of domains that have ruby chromatin in them and that are flanked by aquamarine chromatin, the genome is roughly divided into constitutively active and constitutively inactive zones (Kolesnikova, 2018).

To take into account the tissue-specific features of chromatin organization in salivary gland polytene chromosomes, the morphology of polytene chromosomes, an extensive pool of 'experimental cytology' data, and data on gene expression in salivary glands were examined and, whenever deemed necessary, corrections were introduced into initial predictions. The results of this study revealed that this approach works well (Kolesnikova, 2018).

Yet another unique feature of the four-state chromatin model is that it reveals the chromatin type (specifically, aquamarine) that is enriched with interband-specific proteins; no other models of clustering chromatin proteins can do this. According to the most recent high-resolution Hi-C data from embryos, TAD boundaries correspond with high resolution to polytene chromosome interbands, whereas black and gray bands are the visualization of topological domains with different types of DNA folding. Thus, the four-state chromatin model allows the boundaries of physical (not epigenetic) domains to be found. The work by Hou (2012) clearly indicates that these boundaries are not always the same (Kolesnikova, 2018).

The choice of aquamarine chromatin as potential band boundaries is supported well by comparing coordinates with the distribution of SUUR and H3K27me3 on polytene chromosomes: these two are markers of black bands and display sharp changes on their boundaries. Thus, the approach used in the current work can be conveniently used to identify the coordinates of specific polytene-chromosome black bands with high accuracy. For most black bands, accuracy of 2-10 kb is attainable (Kolesnikova, 2018).

With the band coordinates inferred, a detailed comparison was performed of replication profiles from diploid cells and replication patterns observed in polytene chromosomes and analyzed the properties of black bands (Kolesnikova, 2018).

A considerable degree of similarity in replication timing has been demonstrated between salivary gland polytene chromosomes and diploid cells. In both object types, the zones between black bands correspond to early replication initiation zones. This result is consistent with the observation that most ORC2-binding sites are in aquamarine chromatin corresponding to interbands. Ruby-containing polytene chromosome bands (Rb-bands) in different cell types have a U-shaped replication profile, which implies that replication in them proceeds from the boundaries to the center, leading to a local delay in replication completion, this delay being proportional to the band length (Kolesnikova, 2018).

The averaged boundary replication profiles in INTs that were built from previously published cell culture data are consistent with the prediction that very few sites within the intervals between rb-bands initiate replication in each replication cycle. The typical size of replicons, 80 kb, originating from the early replication initiation zone (data from cell cultures) fits this model well too. Analysis of stretched DNA fibers in D. nasuta polytene chromosomes has revealed that the replicons initiated in the early S phase are each 64 μm in size on average, which should amount to more than 120 kb. The fact that the replicons are that long provides further support to the hypothesis that the replication origins fired during one cycle in a particular DNA molecule should be well spaced. While analyzing replication in partially denatured polytene chromosomes, researchers observed temporal and spatial asynchrony in replication initiation in parallel fibers and proposed that this asynchrony is one of the main reasons for continuous labeling in polytene chromosomes. Although these data come from a Drosophila species irrelevant to this study and the typical sizes and genomic distances may be different to some extent, it is proposed that the organization of replication in polytene chromosomes is conserved across Drosophila species (Kolesnikova, 2018).

Replication patterns change in polytene chromosomes, and these patterns are linked to events in DNA sequences. At the beginning of the S phase, replication is initiated in INTs, which may contain a large number of potential replication initiation sites. In each DNA strand, an initiation event occurs only once per INT in a random interband. Initiation events in different interbands can occur in asynchrony, either in different INTs or in the same INT on the parallel DNA strands of a polytene chromosome. Replication forks move through INTs in the opposite directions from the site of replication initiation and eventually enter the nearest rb-bands. After all INTs have completed replication, the replication fork should be detectable only in rb-bands. This situation is consistent with the observed inverted PCNA pattern, when all black bands produce the signal that the intervals do not (Kolesnikova, 2018).

By analysis of stretched DNA fibers in D. nasuta polytene chromosomes, it has been demonstrated that the rates of replication fork movement in polytene chromosomes during the late S phase are on average one-tenth of those in the early S phase. The authors believe that upon entering polytene chromosome rb-bands, replication forks slow down. That is why, although some rb-bands are shorter than the flanking intervals, all 'black' bands undergoing replication at once. Replication in these bands goes on until the forks moving toward each other meet. In the longest rb-bands, forks fail to meet before the end of the S phase, leading to under-replication. In D. melanogaster, replication rates depend on SUUR. This study demonstrated that all rb-bands are enriched with SUUR both in salivary gland polytene chromosomes and in diploid cells. According to another study, local artificial tethering of SUUR to an early replicating region of a salivary gland polytene chromosome causes delayed replication there. It can be proposed that this protein plays an important role in delayed replication associated with all rb-bands genome-wide (Kolesnikova, 2018).

Evidence exists that the S phase of the endocycle is quite different from that in diploid cells. The former is distinguished by under-replication of a large part of the genome and low expression of genes involved in replication. The presence of the intra-S checkpoint in salivary gland cells is questionable, and so is activation of any late-firing origins. Nevertheless, the results reveal a substantial similarity in replication timing for the euchromatic arm of the whole chromosome. What underlies this similarity is thought to be the organization of the Drosophila genome. The genome consists of alternations of domains capable of initiating early replication (INTs; the interval between black/ruby bands) and domains with the potential to initiate replication late in the S phase. These late domains vary in size, but seldom are they longer than a few hundred kilobases. In diploid cells, these relatively short domains are replicated by replication forks coming from border origins of replication and complete replication before the classic late S phase, which is when late-firing origins activate. Thus, replication of a large portion of a euchromatic arm is, in the classic sense, early replication. Only the most extended bands and regions of pericentric heterochromatin initiate replication in the late S phase. The question of whether replication initiation events occur in the bands is not easy to answer. Schwaiger (2009) analyzed replication profiles and concluded that extended late-replication zones in cell cultures contain origins initiating replication shortly before the end of the S phase. It can be assumed that the replication origins located in rb-bands do not bind all proteins required for independent initiation of replication, and replication on these origins cannot be initiated before a fork comes from outside; these properties are typical of regions showing a U-shaped replication profile in mammals. The same is suggested by recent studies of the genome-wide distribution of the Mcm2-7 helicase complex in D. melanogaster (Kolesnikova, 2018).

Multiple published comparisons of replication profiles for different tissues of the same organism suggest that each cell type has its own schedule of origin activation. One study on individual IH regions indicates that all the 60 analyzed regions are late replicating in cultured cells, but inside those regions, there are local zones of early replication. After artificial induction of transgene expression in IH, there are also local changes in replication timing (Kolesnikova, 2018).

In cell cultures, early-replication zones within rb-bands can be identified by analysis of the outliers in the boxplots of the averaged boundary replication profile. Among all the rb-bands, only two had early-replication zones spanning them from end to end. It can be theorized that in different tissues, most bands similar in size undergo replication within a similar time interval in the S phase, and gene activation in these bands makes the corresponding fragment of the band earlier replicating (Kolesnikova, 2018).

It can be concluded that the alternation of rb-bands and INTs forms the basis of the pattern of replication timing in D. melanogaster. This organization is conserved in eukaryotes. It has been demonstrated that a substantial portion of the mammalian genome represents the alternation of replication initiation zones, in which early master origins lie, and U-shaped replication zones, in which initiation occurs at virtually random positions and in a cascadelike manner, shaping the profile accordingly. The initiation zones are notable for active transcription and high gene density. The boundaries of these zones correspond to those of topological domains (Kolesnikova, 2018).

IH regions represent a separate fraction of black bands, grossly corresponding to the most extended and late-replicating bands (group LR5). This study demonstrated that all rb-bands, including small ones, share a large number of properties with IH regions. This is direct evidence that among all genomic regions, IH regions do not stand out as some special type of sequences. Genes in any rb-band tend to be expressed in a limited number of tissues and, according to GO analysis, these regions are enriched with tissue-specific genes. By contrast, the intervals between black bands are enriched with genes that are highly expressed in most tissues chosen for analysis here. Each rb-band appears as a combination of repressed chromatin types; however, open chromatin can be found in its boundary regions, pointing to a similarity between bands and TADs. It is confirmed that the boundaries of black bands correspond to those of topologically associating domain or sub-domains. TADs represent a stable level of genome organization during development both in mammals and in Drosophila. It has been demonstrated that the partitioning of genomes into physical domains correlates with gene density and transcription distribution. These features are closely associated with replication timing. That late replication correlates with LADs has been demonstrated in both Drosophila and mammals (Kolesnikova, 2018).

The results of this work suggest that Drosophila polytene chromosomes can serve as vivid visualization of the organization of the eukaryotic genome, which is conserved between Drosophila and mammals. The characteristic pattern of polytene chromosomes -- the compacted black bands alternating with less compact grey bands and interbands -- reflects the partitioning of the Drosophila genome into domains with contrasting properties (Kolesnikova, 2018).

Chromatin conformation and transcriptional activity are permissive regulators of DNA replication initiation in Drosophila

Chromatin structure has emerged as a key contributor to spatial and temporal control over the initiation of DNA replication. Nevertheless, a causal relationship between chromatin structure and replication initiation remains elusive. This study combined histone gene engineering and whole-genome sequencing in Drosophila to determine how perturbing chromatin structure affects replication initiation. Most pericentric heterochromatin was found to remain late replicating in H3K9R mutants, even though H3K9R pericentric heterochromatin is depleted of HP1a, more accessible, and transcriptionally active. These data indicate that HP1a loss, increased chromatin accessibility, and elevated transcription do not result in early replication of heterochromatin. Nevertheless, a small amount of pericentric heterochromatin with increased accessibility replicates earlier in H3K9R mutants. Transcription is de-repressed in these regions of advanced replication, but not in those regions of the H3K9R mutant genome that replicate later, suggesting that transcriptional repression may contribute to late replication. This study also explored relationships among chromatin, transcription, and replication in euchromatin by analyzing H4K16R mutants. In Drosophila, the X Chromosome is upregulated 2-fold and replicates earlier in XY males than it does in XX females. This study found that H4K16R mutation prevents normal male development and abrogates hyper-expression and earlier replication of the male X, consistent with previously established genome-wide correlations between transcription and early replication. By contrast, H4K16R females are viable and fertile, indicating that H4K16 modification is dispensable for genome replication and gene expression (Armstrong, 2018).

Super-resolution microscopy reveals stochastic initiation of replication in Drosophila polytene chromosomes

Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. This study developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. Replication initiation zones were identified where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter (Kolesnikova, 2022).

Dynamic changes in ORC localization and replication fork progression during tissue differentiation

Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. This study exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, sites of ORC binding were found to show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation (Hue, 2018).

Regulatory functions and chromatin loading dynamics of linker histone H1 during endoreplication in Drosophila

Eukaryotic DNA replicates asynchronously, with discrete genomic loci replicating during different stages of S phase. Drosophila larval tissues undergo endoreplication without cell division, and the latest replicating regions occasionally fail to complete endoreplication, resulting in underreplicated domains of polytene chromosomes. This study shows that linker histone H1 is required for the underreplication (UR) phenomenon in Drosophila salivary glands. H1 directly interacts with the Suppressor of UR (SUUR) protein and is required for SUUR binding to chromatin in vivo. These observations implicate H1 as a critical factor in the formation of underreplicated regions and an upstream effector of SUUR. It was also demonstrated that the localization of H1 in chromatin changes profoundly during the endocycle. At the onset of endocycle S (endo-S) phase, H1 is heavily and specifically loaded into late replicating genomic regions and is then redistributed during the course of endoreplication. The data suggest that cell cycle-dependent chromosome occupancy of H1 is governed by several independent processes. In addition to the ubiquitous replication-related disassembly and reassembly of chromatin, H1 is deposited into chromatin through a novel pathway that is replication-independent, rapid, and locus-specific. This cell cycle-directed dynamic localization of H1 in chromatin may play an important role in the regulation of DNA replication timing (Andreyeva, 2017).

This study demonstrated that virtually all major sites of UR throughout the Drosophila genome exhibit a substantial increase in salivary gland DNA copy number upon depletion of the linker histone H1, thus implicating H1 in the regulation of endoreplication. In control knockdown salivary glands, 46 underreplicated domains were identified. While these regions are in general agreement with previous efforts to map underreplicated domains by less sensitive microarray analyses, fewer underreplicated sites were identified than a recent report that used high-throughput sequencing of salivary gland DNA (Yarosh, 2014). Notably, the underreplicated domains that the current analyses failed to detect represent sites with the weakest degree of UR. One possible source of variation is the distinct technical approach that was used compared with Yarosh (2014), as simultaneous sequencing of a nonpolytenized (embryonic) genome as a means to normalize the reads from underrepresented sequences in polytenized tissues (Yarosh, 2014) likely provides additional sensitivity. Another potential explanation could lie in the relative sequencing depth of the respective assays (approximately fourfold lower in the current study), considered crucial for the analyses of next-generation sequencing data. However, this explanation is less likely, as subsampling of the current reads to much lower depths yielded no appreciable difference in the number and location of identified underreplicated sites or the change in copy number upon H1 knockdown (Andreyeva, 2017).

On average, a moderate knockdown of H1 led to an ~50% copy number gain at the center of underreplicated domains in intercalary heterochromatin (IH; large dense bands scattered in euchromatin comprising clusters of repressed genes. The copy number is not restored to the same degree as that in a SuUR genetic mutant. The difference is likely attributable to the incomplete depletion of H1. In fact, in an independent biological validation experiment that resulted in an ~95% depletion of H1, an almost complete restoration of copy number was observed. The observation of an almost complete reversal of UR in cells depleted of H1 (but still wild type for SuUR) strongly suggests an epistatic mechanism of action in which both H1 and SUUR act together in the same biochemical pathway (Andreyeva, 2017).

This study found that H1 and SUUR are also involved in UR of PH. For instance, both the mapped pericentric regions and TE sequences, which are highly abundant in pericentric regions, exhibit an increase of DNA copy number upon H1 knockdown. The SuURES mutation also results in a robust loss of UR at PH, as measured by changes in DNA copy number at TEs. The abrogation of H1 expression gives rise to a somewhat weaker effect on the UR of PH than that of IH, which is consistent with an almost complete elimination of SUUR protein from polytene chromosome arms in salivary glands depleted of H1 by RNAi but the persistence of residual SUUR at their PH. The role of H1 in maintaining the underreplicated state of PH may be relevant to its important regulatory functions in constitutive heterochromatin, where it recruits Su(var)3-9, facilitates H3K9 methylation, and maintains TEs in a transcriptionally repressed state. Recently, it was proposed that TE repression in ovarian somatic cells involves an H3K9 methylation-independent process through recruitment of H1 by Piwi-piRNA complexes, resulting in reduced chromatin accessibility. The current results also implicate UR of TE sequences in polytenized cells as yet another putative mechanism that contributes to regulation of their expression. Interestingly, it was shown previously that double mutants encompassing both the Su(var)3-9 and SuUR mutant alleles exhibit a synthetically increased predominance of novel band-interband structures at PH compared with the mutation of SuUR alone. While the evidence suggests a relationship between UR and transcriptionally repressive epigenetic states, such as H3K9 methylation, the nature of this relationship remains largely speculative (Andreyeva, 2017).

This study demonstrated that SUUR protein physically interacts with H1 in both a complex mixture of whole-cell extracts that contain endogenous native H1 and recombinant purified H1 polypeptides. Furthermore, the particular structural domains of the two proteins were delimited that are required for the interaction. SUUR protein contains several sequence features that have been implicated in regulation of UR and binding to specific proteins. Although SUUR possesses a putative bromodomain, it contains no identifiable DNA-binding domain, so the mechanism that allows SUUR to exhibit a preference for specific genomic underreplicated loci is unknown. The positively charged central region is both necessary and sufficient to interact with heterochromatin protein 1a (HP1a), which suggests a possible involvement of HP1a in tethering SUUR to H3K9me2/3-rich PH. However, the specific localization of SUUR to underreplicated IH, which is not enriched for H3K9me2/3, remains enigmatic. This study now demonstrates that the central region of SUUR is also sufficient for binding directly to H1 in vitro. Considering that the central region of SUUR is essential for the faithful localization of the protein to chromatin in vivo, including underreplicated IH, it seems likely that H1 directly mediates the tethering of SUUR to chromatin in underreplicated regions (Andreyeva, 2017).

The tripartite structure of H1 provides multiple binding interfaces for interacting proteins and thus allows H1 to mediate several biochemically separable functions in vivo. For instance, the globular domain and proximal 25% of the CTD are required for H1 loading into chromatin, while the proximal 75% of the CTD is needed for normal polytene morphology, H3K9 methylation, and physical interactions with Su(var)3-9. This study discovered a previously unknown function for the distal 25% of the H1 CTD, which is shown to be essential for binding to SUUR. Deletion of this region of H1 results in a near-complete loss of the interaction with SUUR. Thus, in addition to its critical functions in heterochromatin structure and activity, the CTD of H1 is likely also important in facilitating UR (Andreyeva, 2017).

One of the most striking findings in this study is the observation that the genomic occupancy of H1 undergoes profound changes during the endoreplication cycle. It also remains largely mutually exclusive with that of DNA polymerase clamp loader PCNA, which is consistent with the observed depletion of H1 in nascent chromatin compared with mature chromatin (Andreyeva, 2017).

H1 is heavily loaded into late replicating loci at the onset of replication (when these loci are silent for replication). Combined, the current observations indicate that the chromosome distribution of H1 during the endocycle is governed by at least three independent processes. Two of them [replication-dependent (RD) eviction of H1 and RD deposition of H1 after the passage of replication fork] are related to the well-recognized obligatory processes of chromatin disassembly and reassembly during replication. The third pathway, which directs early deposition of H1 into late replicating loci, has not been described previously. This process is (1) replication-independent (RI); (2) locus-specific, with a strong preference for late replicating sites; and (3) apparently more rapid than the RD deposition of H1, since very high levels of H1 occupancy are observed in all nuclei immediately after the initiation of endo-S. It is possible that the RI pathway of H1 loading into chromatin is mediated by a selective recruitment of H1 based on epigenetic core histone modification-dependent mechanisms. For instance, mammalian H1.2 was reported to recognize H3K27me3, and this modification is very abundant in IH (Sher et al. 2012) (Andreyeva, 2017).

Also, the RI mechanism for deposition of H1 probably does not involve de novo nucleosome assembly, as H1 is known to exhibit a mutually exclusive distribution with RI core histone variants, and there is no known nuclear process during early S phase that requires core histone turnover. In the future, it will be interesting to further confirm that RI nucleosome assembly does not take place during early replication in salivary gland polytene chromosomes. Finally, the locus-specific RI deposition of H1 in early endo-S chromatin may be conserved in the normal S phase of diploid tissues, and it will require independent experimentation with sorted mitotically dividing cells to confirm this possibility (Andreyeva, 2017).

This study also provides cytological evidence that the functions of H1 and SUUR are biochemically linked. Specifically, it was demonstrated that SUUR localizes to a subset of H1-positive bands and requires H1 for its precise distribution in polytene chromosomes, nuclear localization, and stability in salivary gland cells. These observations implicate H1 as an upstream effector of SUUR functions in vivo and an essential component of the biological pathway that maintains loci of reduced ploidy in polytenized cells. Importantly, this finding adds to a growing list of biochemical partners of H1 that mediate their chromatin-directed functions in an H1-dependent fashion (Andreyeva, 2017).

Interestingly, even a moderate depletion of H1 (to ~30% of normal) results in a complete removal of SUUR from chromosome arms. Thus, H1-dependent localization of SUUR requires high concentrations of the linker histone in chromatin. This conclusion is also consistent with SUUR colocalization with polytene loci that are the most strongly stained for H1. In contrast, elimination of the H3K9me2 mark from polytene spreads requires very extensive depletion of H1, whereas the moderate depletion of H1 does not strongly affect H3K9 dimethylation in the chromocenter or polytene arms. Therefore, the robust effect of even moderate H1 depletion on SUUR localization in chromatin is unlikely to be mediated indirectly through disorganization of heterochromatin structure (Andreyeva, 2017).

Unexpectedly, the cell cycle-dependent temporal pattern of H1 localization is not identical to that of SUUR. In contrast to H1, SUUR protein (1) is only weakly present in IH during early endo-S phase, (2) achieves the maximal occupancy at IH loci only in the late endo-S, and (3) colocalizes with PCNA at certain sites. The observations made in this study and in previous works can be summarized in the following model for H1-mediated regulation of SUUR association with chromatin. The initiation of the deposition of SUUR in chromosomes is strongly dependent on H1. More specifically, SUUR is preferentially localized to chromatin domains that are highly enriched for H1. For instance, the tremendously elevated concentration of H1 in IH of early endo-S cells promotes and nucleates the initiation of deposition of SUUR into these regions. However, the pattern of SUUR occupancy at these sites does not occur temporally in parallel with that of H1. Initially, the exceptionally high abundance of H1 in late replicating loci during early endo-S is not paralleled by a simultaneous comparable increase of SUUR occupancy. Rather, loading of SUUR into these sites lags significantly behind H1 occupancy. Thus, the rate of SUUR localization to H1-rich IH appears to be much slower than that of the RI deposition of H1 into these loci. After the initial recruitment, further loading of SUUR does not require H1, and SUUR continues (in a slower fashion) to accumulate at IH throughout the endo-S phase even when H1-enriched domains dissipate in the course of DNA endoreplication. The additional loading of SUUR in chromatin is likely facilitated by its self-association through dimerization of the N terminus and physical interactions with the replication fork, as proposed previously. In this fashion, SUUR achieves its maximal concentration in IH loci by the late endo-S (Andreyeva, 2017).

This study has demonstrated that H1 has a pivotal function in the establishment of UR of specific IH loci in polytenized salivary gland cells. The findings that H1 interacts directly with SUUR in vitro and is required for SUUR localization to late replicating IH in polytene chromosomes in vivo strongly suggest that the H1-mediated recruitment of SUUR promotes UR by obstructing replication fork progression in its cognate underreplicated loci but does not affect replication origin firing. However, the remarkable temporal pattern of H1 distribution in endoreplicating polytene chromosomes suggests that it may also play a direct SUUR-independent role in regulation of endoreplication. This is especially plausible considering that the temporal distribution patterns of SUUR and H1 are dissimilar (Andreyeva, 2017).

In contrast to the role of SUUR in slowing down the replication fork progression during late endo-S phase, H1 (acting in the absence of SUUR during early endo-S) may function to repress the initiation of endoreplication, as proposed in several studies. DNA-seq analyses also suggest this mechanism. Compared with the relatively smooth, flat profiles of DNA copy numbers in SuURES mutant salivary glands, the profiles in H1-depleted cells exhibit a jagged, uneven appearance, indicative of aberrant local initiation of replication. Unfortunately, the experimental system (cytological analyzes of salivary glands) cannot be used to further confirm this idea. First, an extensive depletion of H1 results in the loss of polytene morphology; second, since the staging of endo-S progression is based on PCNA staining, a spurious activation of ectopic replication origins would result in an incorrect calling of the stage. To further complicate these analyses, polytenized cells are not amenable to other methods of cell cycle staging, such as fluorescence-activated cell sorting (FACS). In the future, it will be important to examine the role of H1 in regulation of DNA replication timing in sorted Drosophila diploid cells (Andreyeva, 2017).

Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR's SNF2 domain, an interaction between SUUR and Rif1 was identified. Rif1 has many roles in DNA metabolism and regulates the replication timing program. Repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in a partially SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. These findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression (Munden, 2018).

The SUUR protein is responsible for promoting underreplication of heterochromatin and many euchromatin regions of the genome. Although SUUR was recently shown to promote underreplication through inhibition of replication fork progression, the underlying molecular mechanism has remained unclear. Through biochemical, genetic, genomic and cytological approaches, this study has found that SUUR recruits Rif1 to replication forks and that Rif1 is responsible for underreplication. This model is supported by several independent lines of evidence. First, SUUR associates with Rif1, and SUUR and Rif1 co-localize at sites of replication. Second, underreplication is dependent on Rif1, although Rif1 mutants have a clear pattern of late replication in endo cycling cells. Third, SUUR localizes to replication forks and heterochromatin in a Rif1 mutant, however, it is unable to inhibit replication fork progression in the absence of Rif1. Fourth, Rif1 controls replication fork progression and phenocopies the effect loss of SUUR function has on replication fork progression. Fifth, SUUR is required for Rif1 localization to replication forks. Critically, using the gene amplification model to separate initiation and elongation of replication, it was shown that Rif1 can affect fork progression without altering the extent of initiation. Based on these observations, this study defines a new function of Rif1 as a regulator of replication fork progression (Munden, 2018).

This work suggests that the SNF2 domain of SUUR is critical for its ability to localize to replication forks. This is based on the observation that deletion of this domain results in a protein that is unable to localize to replication forks, but still localizes to heterochromatin. SUUR has previously been shown to dynamically localize to replication forks during S phase, but constitutively binds to heterochromatin (Kolesnikova, 2013; Nordman, 2014). SUUR associates with HP1 and this interaction occurs between the central region of SUUR and HP1 (Pindyurin, 2008). Therefore, it is speculated that the interaction between SUUR and HP1 is responsible for constitutive SUUR localization to heterochromatin, while a different interaction between the SNF2 domain and a yet to be defined component of the replisome, or replication fork structure itself, recruits SUUR to active replication forks during S phase (Munden, 2018).

Uncoupling of SUUR's ability to associate with replication forks and heterochromatin also provides a new level of mechanistic understanding of underreplication. Overexpression of the C-terminal two-thirds of SUUR is capable of inducing ectopic sites of underreplication. In contrast, overexpression of the SUUR's SNF2 domain, in the presence of endogenous SUUR, suppresses SUUR-mediated underreplication (Kolesnikova, 2005). Together with the data presented in this study, it is suggested that overexpression of the SNF2 domain interferes with recruitment of full-length SUUR to replication forks, by saturating potential SUUR binding sites at the replication fork. Although the C-terminal region of SUUR is necessary to induce underreplication (Kolesnikova, 2005), the C-terminal portion of SUUR remains associated with heterochromatin in the SUURΔSNF construct, but this protein is not sufficient to induce underreplication. It is suggested that at physiological levels, the affinity of SUUR for replication forks is substantially diminished in the absence of the SNF2 domain. This work raises questions about the biological significance of SUUR binding to heterochromatin, since without the SNF2 domain SUUR is still constitutively bound to heterochromatin, yet unable to induce underreplication. Additionally, SUUR dynamically associates with heterochromatin in mitotic cells although heterochromatin is fully replicated (Munden, 2018).

While trying to uncover the molecular mechanism through which SUUR is able to inhibit replication fork progression, this study has uncovered an interaction between SUUR and Rif1. Through subsequent analysis, it was demonstrated that Rif1 has a direct role in copy number control and that Rif1 acts downstream of SUUR in the underreplication process. Although underreplication is largely dependent on SUUR, there are several sites that display a modest degree of underreplication in the absence of SUUR. In a Rif1 mutant, however, these sites are fully replicated and there is no longer any detectable levels of underreplication within any regions of the genome. It is possible that Rif1 is capable of promoting underreplication through a mechanism independent of SUUR. Therefore, it is concluded that Rif1 is a critical factor in driving underreplication (Munden, 2018).

Further emphasizing the critical role Rif1 plays in copy number control, this study has shown that Rif1 acts downstream of SUUR in promoting underreplication. SUUR is still able to associate with chromatin in the absence of Rif1 but is unable to promote underreplication. Underreplicated regions of the genome, including heterochromatin, tend to be late replicating, raising the possibility that changes in replication timing in a Rif1 mutant suppresses underreplication. Rif1 mutant endocycling cells of Drosophila display a cytological pattern of late replication, where heterochromatin is discretely replicated. While Rif1 controls replication timing in Drosophila and is necessary for the onset of late replication at the mid-blastula transition (Seller, 2018), it is argued that the changes in copy number associated with loss of Rif1 function are not solely due to a loss of late replication. This is supported by the clear pattern of late replication of heterochromatin in Rif1 mutant endocycling cells, although heterochromatin appears to be fully replicated in these cells. Previous work in mammalian polyploid cells has shown that underreplication is dependent on Rif1, which was attributed to changes in replication timing (Hannibal, 2016). It is important to note that Rif1-dependent changes in replication timing were not measured in this system and that many genomic regions transition from early to late replication in a Rif1 mutant (Foti, 2016). This work raises the possibility that Rif1 has a direct role in mammalian underreplication through a mechanism similar to that of Drosophila and may not simply be due to indirect changes in replication timing. Future work will be necessary to define the role of mammalian Rif1 in underreplication (Munden, 2018).

This analysis of amplification loci demonstrates that Rif1 controls replication fork progression independently of initiation control, thus demonstrating that Rif1 has a specific effect on replication fork progression. Therefore, this study has uncovered a new role for Rif1 in DNA metabolism as a regulator of replication fork progression and copy number control. Rif1 has been identified as part of the replisome in human cells by nascent chromatin capture, a technique that identifies proteins associated with newly synthesized chromatin. Multiple studies have assessed whether loss of Rif1 function affects replication fork progression in yeast, mouse and human cells, but have come to different conclusions. DNA fiber assays have been used to measure fork progression in these studies and nearly all have shown that Rif1 mutants have a slight increase in replication fork progression, although not always statistically significant. There could be several reasons for these differing results; Rif1 may control replication fork progression in specific genomic regions that may be underrepresented in some assays, Rif1 function could vary among different cell types, or sample sizes may have been too small to reach significance. These observations, taken together with these previous studies, leave open the possibility that Rif1-mediated control of replication fork progression could be an evolutionarily conserved function of Rif1. It is not suggested that Rif1 is constitutively associated with replication forks in all cell types. Rather, Rif1 could be recruited to replication forks at a specific time in S phase, or in specific developmental contexts, to modulate the progression of replication forks and provide an additional layer of regulation of the DNA replication program (Munden, 2018).

How could SUUR and Rif1 function in concert to inhibit replication fork progression? This study has shown that Rif1 retention at replication forks is dependent on SUUR. Additionally, underreplication depends on Rif1's PP1-binding motif, raising the possibility that a Rif1/PP1 complex is necessary to inhibit replication fork progression. Rif1/PP1 dephosphorylates DDK-activated helicases to control replication initiation. Recently, however, DDK-phosphorylated MCM subunits have been shown to be necessary to maintain DNA-unwinding enzyme Cdc45~MCM~GINS (CMG) association and stability of the helicase (Alver, 2017). This result suggests that continued phosphorylation of the helicase is necessary for replication fork progression (Alver, 2017). It is proposed that SUUR recruits Rif1/PP1 to replication forks where it is able to dephosphorylate MCM subunits, ultimately inhibiting replication fork progression. Although this mechanism needs to be tested biochemically, it provides a framework to address the underlying molecular mechanism responsible for controlling DNA copy number and could provide new insight into the mechanism(s) Rif1 employs to regulate replication timing. (Munden, 2018).

The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development

The Pol32 protein is one of the universal subunits of DNA polymerase delta (Pol delta), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. This study characterized Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol delta localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol delta would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32's role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. This study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism (Ji, 2019).

The role of DNA repair genes in radiation-induced adaptive response in Drosophila melanogaster is differential and conditional

Studies in human and mammalian cell cultures have shown that induction of DNA repair mechanisms is required for the formation of stimulation effects of low doses of ionizing radiation, named "hormesis". Nevertheless, the role of cellular defense mechanisms in the formation of radiation-induced hormesis at the level of whole organism remains poorly studied. The aim of this work was to investigate the role of genes involved in different mechanisms and stages of DNA repair in radioadaptive response and radiation hormesis by lifespan parameters in Drosophila melanogaster. Genes were studied that control DNA damage sensing (D-Gadd45, Hus1, mnk), nucleotide excision repair (mei-9, mus210, Mus209), base excision repair (Rrp1), DNA double-stranded break repair by homologous recombination (Brca2, spn-B, okr) and non-homologous end joining (Ku80, WRNexo), and the Mus309 gene that participates in several mechanisms of DNA repair. The obtained results demonstrate that in flies with mutations in studied genes radioadaptive response and radiation hormesis are absent or appear to a lesser extent than in wild-type Canton-S flies. Chronic exposure of gamma-radiation in a low dose during pre-imaginal stages of development leads to an increase in expression of the studied DNA repair genes, which is maintained throughout the lifespan of flies. However, the activation of conditional ubiquitous overexpression of DNA repair genes does not induce resistance to an acute exposure to gamma-radiation and reinforces its negative impact (Koval, 2019).

Epigenetic regulation affects gene amplification in Drosophila development

In Drosophila melanogaster, in response to developmental transcription factors, and by repeated initiation of DNA replication of four chorion genes, ovarian follicle cells, form an onion skin-type structure at the replication origins. The DNA replication machinery is conserved from yeast to humans. Subunits of the origin recognition complex (ORC) is comprised of Orc1, Orc2, and Cdc6 genes. While mutations of Orc1 and Orc2 and not Cdc6 can be lethal, overexpression of these genes lead to female sterility. Ecdysone, is a steroidal prohormone of the major insect molting hormone 20-hydroxyecdysone that in Drosophila, triggers molting, metamorphosis, and oogenesis. To this end, several ecdysone receptor (EcR) binding sites were identified around gene amplification loci. It was also found that H3K4 was trimethylated at chorion gene amplification origins, but not at the act1 locus. Female mutants overexpressing Lsd1 (a dimethyl histone H3K4 demethylase) or Lid (a trimethyl histone H3K4 demethylase), but not a Lid mutant, were sterile. The data suggest that ecdysone signaling determines which origin initiates DNA replication and contributes to the development. Screening strategies using Drosophila offer the opportunity for development of drugs that reduce gene amplification and alter histone modification associated with epigenetic effects (Kohzaki, 2019).

Drosophila Xrcc2 regulates DNA double-strand repair in somatic cells

Genomic integrity is challenged by endo- and exogenous assaults that are combated by highly conserved DNA repair mechanisms. High fidelity recombination repair of DSBs relies on the Rad51 recombinase, aided by several Rad51 paralogs. Only two out of four Rad51 paralogs in Drosophila have been studied so far and both are restricted to meiotic recombination repair. Using CRISPR/Cas9 technology, this study has generated the first X-ray repair cross complementing 2 (xrcc2) null mutant in Drosophila. Like any other Drosophila Rad51 homologue, loss of xrcc2 does not affect fly development. Drosophila xrcc2 - despite a specific expression in ovaries - is not essential for meiotic DSB repair, but supports the process. In contrast, xrcc2 is required for mitotic DNA damage repair: the mutants are highly sensitive towards various genotoxic stressors, including ionizing radiation, which significantly increase mortality. Moreover, loss of xrcc2 provokes chromosome aberrations in mitotic larval neuroblasts under unstressed conditions and enduring chromosomal breaks as well as persistent repair foci after irradiation exposure. Together these results demonstrate that xrcc2 plays a crucial role in combating genotoxic insult by controlling DSB repair in somatic cells of Drosophila (Bayer, 2020)

UVA causes specific mutagenic DNA damage through ROS production, rather than CPD formation, in Drosophila larvae

Evidence is accumulating that ultraviolet A (UVA) plays an important role in photo-carcinogenesis. However, the types of DNA damage involved in the resulting mutations remain unclear. Previously, using Drosophila, it was found that UVA from light-emitting diode (LED-UVA) induces double-strand breaks in DNA through oxidative damage in an oxidative damage-sensitive (urate-null) strain. Recently, it was proposed that cyclobutane pyrimidine dimers (CPDs), which also are induced by UVA irradiation, might play a significant role in the induction of mutations. The present study investigated whether reactive oxygen species (ROS) and CPDs are produced in larval bodies following LED-UVA irradiation. In addition, this study assessed the somatic cell mutation rate in urate-null Drosophila induced by monochromatic UVA irradiation. The production of ROS through LED-UVA irradiation was markedly higher in the urate-null strain than in the wild-type Drosophila. CPDs were detected in the DNA of both of UVA- and UVB-irradiated larvae. The level of CPDs was unexpectedly higher in the wild-type strain than in urate-null flies following UVA irradiation, whereas this parameter was expectedly similar between the urate-null and wild-type Drosophila following UVB irradiation. The somatic cell mutation rate induced by UVA irradiation was higher in the urate-null strain than in the wild-type strain. These results suggest that mutations induced by UVA-specific pathways occur through ROS production, rather than via CPD formation (Negishi, 2023).

The effect of repeat length on Marcal1-dependent single-strand annealing in Drosophila

Proper repair of DNA double strand breaks (DSBs) is essential to maintenance of genomic stability and avoidance of genetic disease. Organisms have many ways of repairing DSBs, including use of homologous sequences through homology-directed repair (HDR). While HDR repair is often error-free, in single-strand annealing (SSA) homologous repeats flanking a DSB are annealed to one another, leading to deletion of one repeat and the intervening sequences. Studies in yeast have shown a relationship between the length of the repeat and SSA efficacy. This study sought to determine the effects of homology length on SSA in Drosophila, as Drosophila uses a different annealing enzyme (Marcal1) than yeast. Using an in vivo SSA assay, it was shown that 50 base pairs (bp) is insufficient to promote SSA and that 500-2000 bp is required for maximum efficiency. Loss of Marcal1 generally followed the same homology length trend as wild-type flies, with SSA frequencies reduced to about a third of wild-type frequencies regardless of homology length. Interestingly, a difference was found in SSA rates between 500 bp homologies that align to the annealing target either nearer or further from the DSB, a phenomenon that may be explained by Marcal1 dynamics. This study gives insights into Marcal1 function and provides important information to guide design of genome engineering strategies that use SSA to integrate linear DNA constructs into a chromosomal DSB (Dewey, 2023).

Predicting recombination suppression outside chromosomal inversions in Drosophila melanogaster using crossover interference theory

The interference hypothesis of recombination suppression proposes heterozygous inversion breakpoints possess chiasma-like properties such that recombination suppression extends from these breakpoints in a process analogous to crossover interference. This hypothesis is qualitatively consistent with chromosome-wide patterns of recombination suppression extending to both inverted and uninverted regions of the chromosome. The present study generated quantitative predictions for this hypothesis using a probabilistic model of crossover interference with gamma-distributed inter-event distances. These predictions were then tested with experimental genetic data (>40,000 meioses) on crossing-over in intervals that are external and adjacent to four common inversions of Drosophila melanogaster. The crossover interference model accurately predicted the partially suppressed recombination rates in euchromatic intervals outside inverted regions. Furthermore, assuming interference does not extend across centromeres dramatically improved model fit and partially accounted for excess recombination observed in pericentromeric intervals. Finally, inversions with breakpoints closest to the centromere had the greatest excess of recombination in pericentromeric intervals, an observation that is consistent with negative crossover interference previously documented near Drosophila melanogaster centromeres. In conclusion, the experimental data support the interference hypothesis of recombination suppression, validate a mathematical framework for integrating distance-dependent effects of structural heterozygosity on crossover distribution, and highlight the need for improved modeling of crossover interference in pericentromeric regions (Koury, 2023).

Alternative end-joining results in smaller deletions in heterochromatin relative to euchromatin

Homologous recombination (HR) repair is uniquely regulated in the ericentromeric heterochromatin to enable 'safe' repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. This study induced DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR- white reporter and I-SceI expression in spermatogonia of flies. Using this approach, higher frequency of HR repair is detected in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin (Miller, 2023).

HR repair pathway plays a crucial role in maintaining neural stem cell fate under irradiation stress

Environmental stress can cause mutation or genomic instability in stem cells which, in some cases, leads to tumorigenesis. Mechanisms to monitor and eliminate these mutant stem cells remain elusive. Using the Drosophila larval brain as a model, this study shows that X-ray irradiation (IR) at the early larval stage leads to accumulation of nuclear Prospero (Pros), resulting in premature differentiation of neural stem cells (neuroblasts, NBs). Through NB-specific RNAi screenings, the Mre11-Rad50-Nbs1 complex and the homologous recombination (HR) repair pathway, rather than non-homologous end-joining pathway that plays, a dominant role in the maintenance of NBs under IR stress. The DNA damage sensor ATR/mei-41 is shown to act to prevent IR-induced nuclear Pros in a WRNexo-dependent manner. The accumulation of nuclear Pros in NBs under IR stress, leads to NB cell fate termination, rather than resulting in mutant cell proliferation. This study reveals an emerging mechanism for the HR repair pathway in maintaining neural stem cell fate under irradiation stress (Xu, 2023).

Senescent cells and macrophages cooperate through a multi-kinase signaling network to promote intestinal transformation in Drosophila

Cellular senescence is a conserved biological process essential for embryonic development, tissue remodeling, repair, and a key regulator of aging. Senescence also plays a crucial role in cancer, though this role can be tumor-suppressive or tumor-promoting, depending on the genetic context and the microenvironment. The highly heterogeneous, dynamic, and context-dependent nature of senescence-associated features and the relatively small numbers of senescent cells in tissues makes in vivo mechanistic studies of senescence challenging. As a result, which senescence-associated features are observed in which disease contexts and how they contribute to disease phenotypes remain largely unknown. Similarly, the specific mechanisms by which various senescence-inducing signals are integrated in vivo to induce senescence and why some cells become senescent while their immediate neighbors do not are unclear. This study identified a small number of cells that exhibit multiple features of senescence in a genetically complex model of intestinal transformation established in the developing Drosophila larval hindgut epithelium. These cells emerge in response to concurrent activation of AKT, JNK, and DNA damage response pathways within transformed tissue. Eliminating senescent cells, genetically or by treatment with senolytic compounds, reduces overgrowth and improves survival. This tumor-promoting role is mediated by Drosophila macrophages recruited to the transformed tissue by senescent cells, which results in non-autonomous activation of JNK signaling within the transformed epithelium. These findings emphasize complex cell-cell interactions underlying epithelial transformation and identify senescent cell-macrophage interactions as a potential druggable node in cancer. It is concluded that interactions between transformed senescent cells and macrophages drive tumorigenesis (Datta, 2023).

Ataxia-associated DNA repair genes protect the Drosophila mushroom body and locomotor function against glutamate signaling-associated damage

The precise control of motor movements is of fundamental importance to all behaviors in the animal kingdom. Efficient motor behavior depends on dedicated neuronal circuits - such as those in the cerebellum - that are controlled by extensive genetic programs. Autosomal recessive cerebellar ataxias (ARCAs) provide a valuable entry point into how interactions between genetic programs maintain cerebellar motor circuits. Previous work identified a striking enrichment of DNA repair genes in ARCAs. How dysfunction of ARCA-associated DNA repair genes leads to preferential cerebellar dysfunction and impaired motor function is however unknown. This study used Drosophila to characterize the function of ARCA-associated DNA repair genes in the mushroom body (MB). The MB was shown to be required for efficient startle-induced and spontaneous motor behaviors. Inhibition of synaptic transmission and loss-of-function of ARCA-associated DNA repair genes in the MB affected motor behavior. These motor deficits correlated with increased levels of MB DNA damage, MB Kenyon cell apoptosis and/or alterations in MB morphology. It was further shown that expression of genes involved in glutamate signaling pathways are highly, specifically, and persistently elevated in the postnatal human cerebellum. Manipulation of glutamate signaling in the MB induced motor defects, Kenyon cell DNA damage and apoptosis. Importantly, pharmacological reduction of glutamate signaling in the ARCA DNA repair models rescued the identified motor deficits, suggesting a role for aberrant glutamate signaling in ARCA-DNA repair disorders. In conclusion, the data highlight the importance of ARCA-associated DNA repair genes and glutamate signaling pathways to the cerebellum, the Drosophila MB and motor behavior. It is proposed that glutamate signaling may confer preferential cerebellar vulnerability in ARCA-associated DNA repair disorders (Eidhof, 2023).

Rif1 Functions in a Tissue-Specific Manner To Control Replication Timing Through Its PP1-binding Motif

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early and late replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, this study used the powerful developmental systems available in Drosophila melanogaster. RT profiles were generated and compared from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. The results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, it was demonstrated that the RT differences that were observed in all cases are largely independent of transcriptional differences. A genetic approach was employed in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. The results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, these data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs (Armstrong, 2020).

Flying high-muscle-specific underreplication in Drosophila

Drosophila underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the Drosophila salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. To address this problem, flow cytometry was used to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, it was found that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control (Johnston, 2020).

Effects of Mutations in the Drosophila melanogaster Rif1 Gene on the Replication and Underreplication of Pericentromeric Heterochromatin in Salivary Gland Polytene Chromosomes

In Drosophila salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations SuUR(ES) and Su(var)3-9(06) results in the polytenization of a substantial fraction of unique and moderately repeated sequences but has almost no effect on satellite DNA replication. The Rap1 interacting factor 1 (Rif1) protein is a conserved regulator of replication timing, and in Drosophila, it affects underreplication in polytene chromosomes. This study compared the morphology of pericentromeric regions and labeling patterns of in situ hybridization of heterochromatin-specific DNA probes between wild-type salivary gland polytene chromosomes and the chromosomes of Rif1 mutants and SuUR Su(var)3-9(06) double mutants. Despite general similarities, heterochromatin zones exist that are polytenized only in the Rif1 mutants, and there are zones that are under specific control of Su(var)3-9. In the Rif1 mutants, additional polytenization was found of the largest blocks of satellite DNA (in particular, satellite 1.688 of chromosome X and simple satellites in chromosomes X and 4) as well as partial polytenization of chromosome Y. Data on pulsed incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into polytene chromosomes indicated that in the Rif1 mutants, just as in the wild type, most of the heterochromatin becomes replicated during the late S phase. Nevertheless, a significantly increased number of heterochromatin replicons was noted. These results suggest that Rif1 regulates the activation probability of heterochromatic origins in the satellite DNA region (Kolesnikova, 2020).

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previous work has demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, this study showed that SUUR and Rif1 have differential roles in controlling UR and RT. The findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. This work provides new insight into the process of UR and its links to RT (Das, 2021).

Temporal control of late replication and coordination of origin firing by self-stabilizing Rif1-PP1 hubs in Drosophila

In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the midblastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. This study shows that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a “community effect” counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. A model is proposed in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins (Cho, 2022).

During a typical metazoan cell cycle, large genomic domains initiate their replication at distinct times in S phase. Cytological studies over 60 y ago revealed that DNA sequences in the compacted heterochromatin replicate later in S phase compared to euchromatin. These early studies and recent detailed analyses revealed a complex program among late replicating domains, in which different domains initiate replication with a specific delay. Execution of this stereotyped schedule occupies much of the S phase and must finish before mitosis. Despite recent advances in genomic methods for profiling global replication timing, the basis of the timing control is not yet solved, and how multiple origins are coordinated to fire together especially within repetitive DNA sequences is not known (Cho, 2022).

The Drosophila embryo offers a unique setting in which to examine the control of temporal programing of replication. In the earliest nuclear division cycles, there is no late replication, closely spaced origins throughout the genome initiate replication rapidly at the beginning of interphase, and their simultaneous action results in an extraordinarily short S phase (3.5 min). Late replication is developmentally introduced during the synchronous blastoderm nuclear division cycles, first influencing pericentric satellite sequences that form a major part of metazoan genomes (over 30% in Drosophila). Individual blocks of satellite DNA are typically several megabase pairs in length, each composed of a different simple repetitive sequence. During the 14th cell cycle at the midblastula transition (MBT), the ∼6,000 cells of the entire embryo progress synchronously through a temporal program in which the different satellites are replicated with distinctive delays (4), dramatically extending the duration of S phase (Cho, 2022).

The initial onset of late replication during development provides a simplified context in which to define its mechanism, because numerous complex features associated with replication timing have not yet been introduced. For example, chromatin states can have major impacts on replication timing. Consistent with this, late-replicating satellite sequences are usually heterochromatic, carrying the canonical molecular marks of constitutive heterochromatin (histone H3 lysine 9 methylation and HP1). During initial Drosophila embryogenesis, the satellites lack significant levels of these marks, and they replicate in sync with the rest of the genome. Surprisingly, the introduction of the delays in replication to the satellite sequences precedes a major wave of heterochromatin maturation in the blastoderm embryo. Furthermore, in a Rif1 null mutant (Rif1KO), the S phase of cycle 14 is significantly shorter, and the late replication of satellite sequences is largely absent even though HP1 recruitment appears normal. Thus, a Rif1-dependent program bears virtually full responsibility for the S-phase program at the MBT (Cho, 2022).

Rif1 is a multifunctional protein with an evolutionarily conserved role in regulating global replication timing. In species from yeast to mammals, mutation or depletion of Rif1 disrupts genome-wide replication timing. Studies in a variety of systems revealed several aspects of Rif1 function. Yeast Rif1 associates with late origins, while the Rif1 of both Drosophila and mammals binds broadly within large late-replicating domains. Rif1 has a conserved motif for interacting with protein phosphatase 1 (PP1), and mutations in the PP1-interacting motifs lead to hyperphosphorylation of MCM helicase in the prereplicative complex (pre-RC) and the disruption of global replication timing. Rif1 itself also harbors many sites recognized by S-phase kinases, including CDK and DDK, near its PP1-interacting motifs. In yeast, both a Rif1 mutant with phosphomimetic changes at these phosphorylation sites and a null mutation of Rif1 partially restore the growth defect of DDK mutants. These data suggest an interplay of Rif1 and DDK, wherein DDK acts first upstream of Rif1 phosphorylating it to disrupt its interaction with PP1, thus lowering the threshold of S-phase kinase activities required for origin firing. Second, DDK acts downstream to directly phosphorylate pre-RC and trigger origin firing. However, how these various features of Rif1 and DDK functions are integrated over large genomic regions to provide a domain-level control of replication timing remains elusive (Cho, 2022).

Studies in flies indicate that Rif1 has adopted a developmental role in governing the onset of the late replication program described above. During the early embryonic cell cycles, high Cdk1 and DDK activities jointly inhibit maternally deposited Rif1, promoting synchronous firing of origins throughout the whole genome to ensure completion of DNA replication during the short interphases. As the cell cycle begins to slow and oscillations in Cdk1 activity emerge, a transient Rif1-dependent delay in the replication of satellite sequences slightly prolongs S phase. When the embryo enters the MBT in cycle 14, abrupt down-regulation of Cdk1 more fully derepresses Rif1, which accumulates in semistable foci (hubs) at satellite DNA loci. High-resolution live microscopy reveals that different Rif1 hubs disperse abruptly at distinct times, followed by proliferating cell nuclear antigen (PCNA) recruitment as the underlying sequences replicate. Mutated Rif1 that is nonphosphorylatable at a cluster of CDK/DDK sites fails to dissociate from satellite DNA and dominantly blocks the completion of satellite DNA replication before mitosis. Conversely, ectopically increasing CDK activity in cycle 14 shortens the persistence of endogenous Rif1 foci and advances the replication program. These findings suggest that each Rif1 hub maintains a local nuclear microenvironment high in Rif1-recruited PP1 that inhibits DNA replication, and that kinase-dependent dispersal of Rif1 hubs is required to initiate the replication of satellite sequences. If it were understood what coordinates Rif1 dispersal throughout the large Rif1 hubs, this model could explain how firing of clusters of the underlying origins is coordinated and how replication of different satellites occurs at distinct times. However, the precise mechanisms controlling the dynamics of Rif1 hubs remain unclear (Cho, 2022).

Since Rif1 can recruit PP1 and form phosphatase-rich domains in the nucleus, it was hypothesized that localized PP1 counteracts kinase-induced Rif1 dissociation so that the Rif1 hubs are self-stabilizing. If this self-stabilization is communicated within each hub, a breakdown in self-stabilization would lead to a concerted collapse of the entire hub and allow origin firing throughout the associated satellite sequence. The current findings indicate that the opposing actions of phosphatase and kinase combined with communication within the hubs create a switch in which a large phosphatase-rich domain is stable until kinase activity overwhelms the phosphatase. It is proposed that for large late-replicating regions of the genome, recruitment of Rif1-PP1 creates a new upstream point of DDK-dependent regulation in which DDK triggers the collapse of the phosphatase-rich domain to create a permissive environment for kinase-induced firing of all previously repressed origins (Cho, 2022).

This study has investigated the mechanisms that control the timing of Rif1 foci dispersal from satellite sequences, which dictates the onset of late replication in the MBT embryo. Rif1-recruited PP1 was demonstrated to mediate self-stabilization of Rif1 hubs, while the S-phase kinase DDK opposes PP1 action and triggers the dispersal of Rif1 hubs. A model is proposed in which the firing of late origins is primarily controlled by a de-repression step upstream of the activation of the pre-RC. In this model, hubs of Rif1 create domains of locally high PP1 that prevent kinase activation of underlying pre-RCs. However, a changing balance of local phosphatase and kinase levels leads to the abrupt destabilization of different Rif1 hubs at distinct times (see A model for the multiple actions of PP1 in stabilizing Rif1 hubs.). This alleviates PP1 inhibition of hub-associated origins at specific times to trigger replication of the different satellites at different times. While this simple model appears sufficient to explain the late replication at its initial onset in the early Drosophila embryo, numerous other factors impact the replication program at later stages when chromatin acquires more complex features. Nonetheless, as is discussed below, the simplicity of the process in this biological context offers some insights into the more enigmatic aspects of late replication, and perhaps suggests a flexible regulatory paradigm that might be used in diverse contexts (Cho, 2022).

While the mechanism is unknown, it has long been clear that large domains of the genome behave as timing units, and that the numerous origins within such domains fire coordinately if not synchronously. The hub model of late replication control in the early embryo can explain how the firing of numerous origins within megabase pairs of satellite sequences can be coordinated in late S phase. Each Rif1 hub is associated with a locus of repetitive satellite sequence (10). Coordinated dispersal of a Rif1 hub will convert the subnuclear compartment from one restricting kinase actions to a permissive one, allowing the activation of pre-RCs throughout the associated chromatin domain. It was previously unclear what leads to the coordinated dispersal of these large hubs. This study shows that a mutant Rif1 that is deficient in binding PP1 cannot form stable hubs on its own, but it joins wild-type Rif1 in semistable hubs. Importantly, the mutant and wild-type Rif1 disperse together, showing that they respond equally to the property of the domain. It is suggested that Rif1-bound PP1 can act in trans to stabilize nearby Rif1-PP1 and that the propagation of this action coordinates the behavior of Rif1 across the entire hub (Cho, 2022).

The contribution of PP1 to the self-stabilization of Rif1 hubs might be mediated by feedback at multiple levels): 1) PP1 might activate itself by removing inhibitory phosphorylation catalyzed by Cdk1; 2) It could reverse Cdk1/DDK-mediated phosphorylation of Rif1 that disrupts PP1-recruitment; 3) It could reverse phosphorylation of Rif1 that disrupts Rif1 chromatin association; or 4) In a circuitous pathway, if the firing of origins were to promote Rif1 dissociation, PP1-dependent suppression of origin firing would stabilize the hubs. Any or all the above actions could reinforce the stability of Rif1-PP1 hubs, perhaps making different contributions in different situations and different organisms. However, regardless of the feedback route, a local dominance of PP1 will stabilize the Rif1 hubs, and rising kinase activity could erode this dominance of PP1. Upon reaching a tipping point, the local PP1 would no longer successfully stabilize the Rif1 hub, and S-phase kinases would then trigger complete dispersion and allow replication of the underlying chromatin (Cho, 2022).

A potential ability of origin firing to feedback and destabilize Rif1 hubs might explain observations in other organisms suggesting that the level of a variety of replication initiation factors can influence replication timing. For example, overexpression of four replication factors including a DDK subunit in the Xenopus embryo shortens the S phase at the MBT. While this has been interpreted as evidence for governance of replication timing by limitation for these factors, the effect may be indirect if overproduction of these factors overrides Rif1 suppression of pre-RC activation to advance the replication of late replicating regions as is seen in the fly embryo (Cho, 2022).

Importantly, the replication defects resulting from Cdc7 knockdown or inhibition of Cdc7, are suppressed in a Rif1 null mutant background. This shows that the level of DDK activity required to reverse or override Rif1 suppression of pre-RC activation is greater than the level needed for direct pre-RC activation. Thus, in a scenario in which rising levels of DDK during S-phase 14 act as a timer, genomic domains associated with Rif1 hubs would fail to replicate until DDK reached the high level required to destabilize the hub. This argues that replication timing depends on the threshold for derepression of the domain rather than on distinct thresholds for firing individual pre-RCs. It is therefore suggested that the timing of late replication is governed at the level of the upstream derepression step in Drosophila embryos, in contrast to the model proposed for other organisms according to which activation of pre-RCs are directly limited by availability of DDK and other replication factors. To produce the distinct temporal program of replication of different satellites, the current model requires domain-specific distinctions in the threshold for hub dispersal. Different satellite loci that are composed of a common repeat sequence replicate at the same time, while satellites composed of different sequences replicate at distinct times. This leads to a proposel that the sequence of repeats influences, likely indirectly, the threshold for Rif1 hub dispersal and the timing of replication (Cho, 2022).

The possible generality of the circuitry this study has defined in the cycle 14 Drosophila embryo can be considered in various ways. Focusing directly on Rif1 involvement in late replication, it is clear that Rif1 does not bare full responsibility for late replication at other stages. Nonetheless, a dosage-dependent function of Rif1 in controlling replication timing is also observed in Drosophila follicle cells during their mitotic cycles. Furthermore, in mammalian cells, ChIP-seq and microscopy showed that Rif1 interacts with large late-replicating domains but, as was seen in cycle 14 embryos, is absent once onset of replication of the underlying chromatin is detected. It is suggested that the mechanism described in this study will be one of multiple contributors to replication timing control in other biological contexts, and it is likely to be the major mode of replication timing in the rapid cycles of externally developing animal embryos (Cho, 2022).

Rif1 has other regulatory roles beyond timing control of pre-RC activation. In the follicle cells of Drosophila egg chambers, Rif1 is recruited to specialized replication forks during chorion gene amplification where it suppresses fork progression. While this action of Rif1 is dependent on its ability to associate with PP1, other possible parallels to the mechanism described in this study are not evident. Rif1 also regulates biological processes beyond replication. It is recruited to regions of DNA damage in mammals as well as to the telomeres in yeast where it has regulatory roles involving distinct interactions. Thus, Rif1 recruitment appears to trigger alternative regulatory pathways in different circumstances (Cho, 2022).

Despite the evident diversity of biological regulation, the capacity of Rif1 to form local membraneless compartments dominated by phosphatase and to abruptly dissolve in response to kinase levels might be an example of a group of flexible regulatory strategies. Many important regulatory events, such as phosphorylation, acetylation, and ubiquitination, are countered by reverse reactions. Various processes, notably the formation of liquid-like condensates, promote local accumulation of proteins. Accumulations of proteins that promote or oppose regulatory modifications could control major regulatory pathways. Furthermore, since protein accumulations could be stabilized or destabilized by the modifications they regulate, a feedback mechanism could control the formation and destabilization of a compartment to give precise spatiotemporal control, as exemplified by the behavior of the Rif1 hubs in the cycle 14 Drosophila embryo (Cho, 2022).

Nucleoporins facilitate ORC loading onto chromatin

The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. This study perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, it was found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. This work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation (Richards, 2022).

Molecular determinants of phase separation for Drosophila DNA replication licensing factors

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, it was shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. In this study, using D. melanogaster (Dm) Cdt1 as a model initiation factor, it was shown that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori (Parker, 2021).

Previous work has shown that several factors used to initiate DNA replication in metazoans - in particular the Orc1 subunit of ORC, as well as Cdc6 and Cdt1 - possess an N-terminal IDR that promotes protein phase separation upon binding DNA. When these initiator proteins are combined and DNA is added, they form comingled condensates that exclude noncognate phase-separating proteins and are active for the ATP-dependent recruitment of the Mcm2-7 helicase. The biophysical mechanisms underlying LLPS by replication initiators have remained unknown (Parker, 2021).

This study used Drosophila Cdt1 as a model system to dissect the molecular basis for DNA-dependent phase separation by initiation factors. These studies show that this condensation mechanism has several distinctive features compared to other proteins characterized to date that also undergo LLPS. Two types of intermolecular interactions synergize with each other to drive phase separation by Cdt1. One set of interactions occur between Cdt1 and DNA. These associations appear to be nonspecific and electrostatic in nature, with DNA serving as a polyanion that bridges the cationic IDRs of multiple Cdt1 molecules. A second set of interactions that occur between the IDRs of different Cdt1 protomers were also identified; these appear to be salt-insensitive and primarily hydrophobic in nature. Importantly, it was found that inter-IDR interactions can drive Cdt1, ORC, and Cdc6 LLPS in the absence of DNA. When these interactions in Cdt1 were abolished, DNA is insufficient to induce phase separation. Thus, inter-IDR interactions provide the primary force behind initiator LLPS, with electrostatic DNA-bridging interactions contributing an additional adhesive force (Parker, 2021).

In the absence of a crowding reagent, DNA is required to induce initiator phase separation at physiological salt and protein concentrations. This dependency suggests that DNA serves as an essential nucleating factor that determines where Cdt1 can form condensates, ensuring that initiator LLPS is restricted to a chromatin context. Interestingly, other anionic biopolymers, such as RNA and heparin, are also capable of inducing phase separation by Cdt1. This type of scaffold 'promiscuity' has been observed in noninitiator systems that undergo LLPS, such as for the nucleolar protein fibrillarin. Given that the cytosol contains multiple anionic biopolymers, future work will need to answer how replication initiators are specifically targeted to chromatin, a question that is similarly relevant for RNA-dependent cellular bodies. The selection of an appropriate nucleic acid scaffold across different phase-separating systems is likely to rely on both general mechanisms, such as the sequestration of alternative, inappropriate scaffolds by proteins with different or nonexistent LLPS properties, and by system-specific mechanisms, such as a requirement for particular sequences or secondary structure (Parker, 2021).

A growing body of work has demonstrated the importance of aromatic residues in driving protein phase separation, along with a related role for π-π and π-cation interactions. In parallel, many studies have shown that condensates formed through aromatic residue interactions are readily dissolved by 1,6-HD. Cdt1 LLPS is both resistant to treatment with 1,6-HD and does not rely on aromatic residues within its IDR for phase separation. ORC and Cdc6 phase separation is also resistant to 1,6-HD. Although this study has not explicitly investigated the role of aromatic residues within these proteins, it is noted that the fraction aromatic residues of Orc1 (0.019) and Cdc6 (0.012) is lower than that of Cdt1 (0.027), suggesting that aromaticity is dispensable for LLPS by replication initiation factors in general. Similarly, charged residues have an established role in facilitating homomeric inter-IDR interactions that lead to phase separation, yet the inter-IDR interactions that drive initiator phase separation are salt insensitive (Parker, 2021).

Instead of relying on aromatic-mediated π-π/π-cation interactions or charge-charge interactions, the data show that interactions between Cdt1 IDRs are instead facilitated by branched hydrophobic residues. Although hydrophobic sequences have been shown to be important for the phase separation of other factors, such as the extracellular matrix protein elastin and the nephrin intracellular domain (NICD), important mechanistic distinctions exist with respect to metazoan replication initiators. For example, both NICD and Cdt1 bear an identical FCRs (FCR = 31%) and rely on a counterion for phase separation. However, inter-IDR interactions within NICD appear to rely primarily on sequence aromatics (11% aromatic content), with hydrophobic residues playing only a supporting role in the self-association of this protein. By contrast, elastin is more like metazoan replication initiators in that it is relatively devoid of aromatic residues (3.9%) and contains a similar content of branched hydrophobic amino acids that underly phase separation (elastin and Cdt1 fraction-L,I,V = 0.21 and 0.19, respectively); however, elastin also differs from initiators in that it readily forms condensates in the absence of a counterion. Elastin's hydrophobic sequences are additionally contained within highly repetitive 'VPGVG' and 'GLG' sequences, and scrambling or shuffling these motifs negatively impacts elastin phase separation. For their part, initiator IDRs lack such repeats and (for Cdt1 at least) LLPS is insensitive to sequence order. It is speculated that the inter-IDR interactions which occur in initiators are weaker than those of Elastin due to the nonrepetitive, distributed nature of their hydrophobic residues, a property that necessitates additional counterion-mediated interactions to promote Cdt1 self-assembly (Parker, 2021).

Understanding the distinctive molecular interactions of different phase-separating systems is essential for developing in silico approaches for predicting the cellular partitioning of a given IDR from primary sequence alone. Such efforts will ultimately require an understanding of both the specific and general features of condensates' scaffolding components (e.g., repetitive motifs vs. amino acid composition). The present work highlights the importance of the general sequence features of initiator IDRs (as opposed to strict amino acid order) in promoting LLPS. Strikingly, this study found that the Cdt1 IDR can be fully scrambled without losing the ability to form condensates and that condensates formed from the scrambled variant can recruit wild-type Cdt1 into droplets. Regions of NICD can similarly be scrambled without losing the ability to form cellular condensates, as can the LCDs of certain transcription factors without affecting promoter targeting. These studies demonstrate the importance of sequence composition in not only driving phase separation but also in facilitating interactions with partner proteins, and is consistent with recent work showing that proteins with similar functional annotation have disordered sequences with similar, evolutionary conserved physical-chemical features (pI, composition, FCR, kappa, etc.). The observations involving the cocondensation properties of Cdt1, ORC, and Cdc6 - three proteins with IDRs of similar amino acid composition but no direct sequence homology - provide experimental support for this 'like-recruits-like' concept. Future work will need to address whether heteromeric inter-IDR interactions (e.g., Orc1IDR-CdcIDR) are indeed governed strictly by sequence composition or whether linear sequence motifs facilitate specific interactions, as was recently suggested for human Orc1 and Cdc6. Further, while metazoan Cdt1 orthologs have no identifiable sequence identity across their disordered domains, they do have a similar amino acid composition. This led to a prediction that the capacity to phase-separate was likely conserved across metazoan initiators, and this was confirmed for human Cdt1. These predictions were further validated for human Orc1 and Cdc6 with the demonstration that these proteins form condensates in the presence of DNA. These results suggest that certain classes of disordered domains can have conserved functionality in the absence of linear sequence identity (Parker, 2021).

Although amino acid composition is the primary determinant underpinning LLPS by replication initiators, short sequence motifs can nonetheless play a critical role in modulating condensation behavior and controlling biological function. Sequence information within the Orc1 and Cdt1 IDR's is high, and while saltatory leaps seem to have occurred between phyla, the conservation of sequence homology within the Drosophila genus, over millions of years, is remarkable and indicates an evolutionary pressure on sequence order for unknown function. A conserved sequence feature within the IDRs of Orc1, Cdt1, and Cdc6 are CDK phosphorylation sites which are known to regulate replication initiation in vivo. It has been previously shown that the phosphorylation of these sites - which are distributed throughout the initiator IDRs - by CDKs abrogates the ability of the Drosophila initiators to form condensates, and a recent finding shows that phosphorylation impedes inter-IDR interactions between human ORC1 and CDC6 as well. Notably, consensus motifs for CDK-dependent phosphorylation (full site = [S/T]PX[R/K] and minimal site = [S/T]P) are conserved in a majority of metazoan Cdt1s and in all sequenced Drosophilidae Cdt1 orthologs, leading to a prediction that phospho-tunable phase separation is a broadly conserved mechanism for regulating metazoan replication licensing. Given that a scrambled IDR can still support LLPS in vitro through both self-interactions and cross-interactions with a wild-type IDR sequence, it will be useful to determine whether such a construct (with or without the native CDK sequences) can support normal kinetics of chromatin association and MCM recruitment and/or cell viability. Such studies, along with deletion and more targeted mutagenesis efforts, will be important to define how the plasticity of IDR sequences can be fine-tuned to elicit specific timing and partnering responses (Parker, 2021).

Defining the molecular 'grammar' that encodes LLPS in metazoan initiators likewise has implications for understanding the impact of initiator phase separation in vivo. Admittedly, neither this nor a previous study provides unequivocal evidence for initiator phase separation in cells. It is also unclear what function such concentrated initiator assemblies might have, such as improving the kinetics or efficiency of helicase loading (as has suggested, or something else entirely, such as maintaining the appropriate nuclear levels of the licensing factors. It is noted that the system lacks the convenience of identifying initiator condensation morphologically (i.e., by the presence of round cellular foci), as mitotic chromosomes - the stage of initiator assembly - are relatively rigid structures built from a central proteinaceous scaffold through an energy-consuming reaction. Thus, it is predicted that initiators condense on the surface of mitotic chromosomes without altering their shape. Consistently, work in multiple model organisms has revealed a switch-like transition in initiator localization during anaphase, at which point initiators begin to coat chromosomes. It is predicted that phosphorylation-dependent control of initiator self-assembly underlies this behavior and produces a dense, replication-competent zone of chromatin-bound initiators poised to drive the precipitous, genome-wide loading of the Mcm2-7 complex in late mitosis. This moment in the cell cycle is an opportune time to prepare for replication, as conflicts with the transcriptional machinery are minimized and the chromatin substrate is, relative to interphase, uniformly compacted. The identification of Cdt1 mutants that selectively block phase separation, but not DNA binding, affords an opportunity to directly investigate the physical nature of these global initiator interactions in vivo (Parker, 2021).

Beyond helicase loading and licensing, initiator LLPS might also serve to prime replisome assembly. Following helicase loading, Mcm2-7 is phosphorylated by the Dbf4-dependent kinase, a heterodimer of Cdc7 and Dbf4. Intriguingly, the Drosophila homolog of Dbf4, Chiffon, contains multiple IDRs, including a large internal IDR (residues 903-1209) that possesses striking compositional similarity to initiator-type IDRs. The identification of an IDR in Chiffon with compositional similarity to the initiator-type IDRs suggests that such sequences may be present in chromatin-associated factors beyond replication licensing components. Indeed, the capacity to undergo DNA-dependent phase separation may have broad utility for chromatin-localized factors and their respective cellular pathways. Thus, future work will aim to develop algorithms that accurately identify proteins proteome wide that possess disordered domains with compositional homology to initiator IDRs and to understand how these sequences impact protein functional dynamics (Parker, 2021).

In summary, the present work provides a detailed view of the DNA-dependent LLPS mechanism for Drosophila Cdt1. Due to a high level of IDR compositional homology and an ability to form comingled phases, the mechanism describe for Cdt1 phase separation likely extends to the other replication initiation factors, ORC and Cdc6. These studies set the stage for investigating the physiological significance of initiator self-assembly in replication licensing and for identifying other sequences in the proteome that possess IDRs capable of associating with chromatin and initiation factors alike (Parker, 2021).

Drosophila SUMM4 complex couples insulator function and DNA replication control

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underrelicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. This study developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. This findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins (Andreyeva, 2022).

Replication of metazoan genomes occurs according to a highly coordinated spatiotemporal program, where discrete chromosomal regions replicate at distinct times during S phase. The replication program follows the spatial organization of the genome in Megabase-long constant timing regions interspersed by timing transition regions (Marchal, 2019). The spatiotemporal replication program exhibits correlations with genetic activity, epigenetic marks, and features of 3D genome architecture and subnuclear localization. Yet the reasons for these correlations remain obscure. Interestingly, the timing of firing for any individual origin of replication is established during G1 before pre-replicative complexes (pre-RC) are assembled at origins, suggesting a mechanism that involves factors other than the core replication machinery (Andreyeva, 2022).

Most larval tissues of Drosophila melanogaster grow via G-S endoreplication cycles that duplicate DNA without cell division, resulting in polyploidy. Endoreplicated DNA molecules frequently align in register to form giant polytene chromosomes. Importantly, in some cell types, genomic domains corresponding to the latest replicated regions of dividing cells, specifically pericentric (PH) and intercalary (IH) heterochromatin, fail to fully replicate during each endocycle resulting in underreplication (UR). These regions are depleted of sites for binding the Origin of Replication Complex (ORC), and thus, their replication primarily relies on forks progressing from external origins in both dividing and endoreplicating cells, which suggests that both cell types utilize related mechanisms of regulation of late replication. Although cell cycle programs are dissimilar between endoreplicating and mitotically dividing cells, they likely share the components of core biochemical machinery for DNA replication. Thus, underreplication provides a facile readout for late replication initiation and delayed fork progression (Andreyeva, 2022).

The Suppressor of UnderReplication (SuUR) gene is essential for polytene chromosome underreplication in intercalary and pericentric heterochromatin (Belyaeva, 1998). In SuUR mutants, the DNA copy number in underreplicated regions is partially restored to almost reach those for fully polyploidized regions of the genome. SuUR encodes a protein (SUUR) containing a helicase domain with homology to that of the SNF2/SWI2 family. The occupancy of ORC in intercalary and pericentric heterochromatin is not increased in SuUR mutants (Sher, 2012), and, thus, the increased replication of underreplicated regions is likely not due to the firing of additional origins. Rather, SUUR negatively regulates the rate of replication fork progression (Nordman, 2014) by an unknown mechanism. It has been proposed (Posukh, 2015) that retardation of the replisome by SUUR takes place via simultaneous physical association with the components of the fork (e.g., CDC45 and PCNA) and repressive chromatin proteins, such as HP1a (Andreyeva, 2022).

Using a newly developed proteomics approach, this study discovered that SUUR forms a stable stoichiometric complex with a chromatin boundary protein Mod(Mdg4)-67.2. SUUR and Mod(Mdg4)-67.2 together are required for maximal underreplication of intercalary heterochromatin and full activity of the gypsy insulator, thereby implicating insulators in obstructing replisome progression and the control of late DNA replication (Andreyeva, 2022).

This paper presents a facile method, termed MERCI, to rapidly identify subunits of stable native complexes by only partial chromatographic purification. It allows one to circumvent the conventional, rate-limiting approach to purify proteins to apparent homogeneity. Since a multistep FPLC scheme invariably leads to an exponential loss of material, reducing the number of purification steps in the MERCI protocol allows identification of rare complexes, such as SUMM4, which may be present in trace amounts in native sources. On the other hand, MERCI obviates introduction of false-positives frequently associated with tag purification of ectopically expressed targets that render results less reliable. Notably, MERCI is not limited to analyses of known polypeptides since it is readily amenable to fractionation of native factors based on a correlation with their biochemical activities in vitro (Andreyeva, 2022).

Both subunits of SUMM4 contribute to the known functions of gypsy insulator. Although a SuUR mutation decreased the insulator activity, the suppression was universally weaker than that by mod(mdg4)u1. It is possible that SUUR is not absolutely required for the establishment of the insulator. For instance, the loss of SUMM4 may be compensated by the alternative complex of Mod(Mdg4)-67.2. Furthermore, the mod(mdg4)u1 allele is expected to have an antimorphic function since it can mis-localize interacting partner proteins, including SUUR itself. Interestingly, SuUR has been previously characterized as a weak suppressor of variegation of the whitem4h X chromosome inversion allele, which places the white gene near pericentric heterochromatin. In contrast, SuUR mutation enhances variegation in the context of insulated, heterochromatin-positioned white. Therefore, this phenotype is unrelated to the putative Su(var) function of SuUR but, rather, is insulator-dependent (Andreyeva, 2022).

Discovery and analyses of SUMM4 provide a biochemical link between ATP-dependent motor factors and the activity of insulators in the regulation of gene expression and chromatin partitioning. Insulator elements organize the genome into chromatin loops that are involved in the formation of topologically associating domains [TADs]. In mammals, CTCF-dependent loop formation requires ATP-driven motor activity of SMC complex cohesin. In contrast, CTCF and cohesin are thought to be dispensable for chromatin 3D partitioning in Drosophila. Instead, the larger, transcriptionally inactive domains (canonical TADs) are interspersed with smaller active compartmental domains, which themselves represent TAD boundaries (Rowley et al., 2017). It has been proposed that in Drosophila, domain organization does not rely on architectural proteins but is established by transcription-dependent, A-A compartmental (gene-to-gene) interactions. However, Drosophila TAD boundaries are enriched for architectural proteins other than CTCF, and their roles have not been tested in loss-of-function models. Thus, it is possible that in Drosophila, instead of CTCF, the 3D partitioning of the genome is facilitated by another group of insulator proteins, such as Su(Hw) and SUMM4, that together associate with class 3 insulators (Andreyeva, 2022).

Moreover, SUUR may provide the DNA motor function to promote a physical separation of active and inactive loci and help establish chromosome contact domains. It is proposed that within the SUMM4 complex, SUUR utilizes its putative ATP-dependent motor activity to translocate along chromatin strands, thus facilitating the establishment of higher-order structures that isolate promoters from enhancers and stabilize DNA loops/domains to prevent unrestricted heterochromatin encroachment and penetration of replication forks. The translocation model is consistent with observations of an asymmetric, selective occupancy of SUUR away from its initial sites of deposition via Su(Hw)-Mod(Mdg4) binding toward inside of intercalary heterochromatin regions but not outside, which may be facilitated by physical interactions between SUUR and linker histone H1 enriched in intercalary heterochromatin. It has been reported that another Drosophila BTB/POZ domain insulator protein CP190 forms a complex with a DEAD-box helicase Rm62 that contributes to the insulator activity. Thus, ATP-dependent motor proteins may represent an obligatory component of the insulator complex machinery (Andreyeva, 2022).

This work explains previous observations about biological functions of SUUR. For instance, the initial deposition of SUUR and its colocalization with PCNA has been proposed to depend on direct physical interaction with components of the replisome (Kolesnikova, 2013). The model developed in this study indicates that, instead, the apparent colocalization of SUUR with PCNA throughout endo-S phase may be caused by a replication fork retardation at insulator sites. SUUR is deposited in chromosomes as a subunit of SUMM4 complex at thousands of loci by tethering via Mod(Mdg4)-Su(Hw) interactions. As replication forks progress through the genome, they encounter insulator complexes where replication machinery pauses for various periods of time before resolving the obstacle. Thus, the increased co-residence time of PCNA and SUUR manifests cytologically as their partial colocalization. With the progression of endo-S phase, some of the SUMM4 insulator complexes are evicted and, thus, the number of SUUR-positive loci is decreased, until eventually the replication fork encounters nearly completely impenetrable insulators demarcating the underreplicated domain boundaries (Andreyeva, 2022).

This mechanism is especially plausible given that boundaries of intercalary heterochromatin loci very frequently encompass multiple, densely clustered Su(Hw) binding sites. This study examined the data from genome-wide proteomic analyses for Su(Hw) and SUUR performed by DamID in Kc167 cells. Strikingly, Su(Hw) DamID-measured occupancy does not exhibit a discrete pattern expected of a DNA-binding factor. Instead, it appears broadly dispersed, together with SUUR, up to tens of kbp away from mapped Su(Hw) binding sites. Interestingly, when hidden Markov modeling was applied to the DamID data, Su(Hw), Mod(Mdg4)-67.2, and SUUR occupancies were found to strongly correlate genome-wide in a novel chromatin form ('malachite') that frequently demarcates the boundaries of intercalary heterochromatin. These observations strongly corroborate the translocation model for the mechanism of action of SUMM4. According to this model, upon tethering to DNA-bound Su(Hw), SUMM4 traverses the underreplicated region, which helps to separate it in a contact domain. As DNA within the underreplicated region is tracked by SUUR, it is brought into a transient close proximity with both SUMM4 and the associated Su(Hw) protein, which is detected by DamID (or ChIP) as an expanded occupancy pattern (Andreyeva, 2022).

The deceleration of SUUR-bound replication forks was also invoked as an explanation for the apparent role of SUUR in the establishment of epigenetic marking of intercalary heterochromatin (Posukh, 2015). It is proposed that global epigenetic modifications observed in the SuUR mutant likely do not directly arise from derepression of the replisome as suggested but, rather, result from the coordinate insulator-dependent regulatory functions of SUUR in both the establishment of a chromatin barrier and DNA replication control (Andreyeva, 2022).

This work demonstrates for the first time that insulator complexes assembled on chromatin can attenuate the extent of replication in discrete regions of the salivary gland polyploid genome. Despite distinct cell cycle programs in dividing and endoreplicating cells, the core biochemical composition of replisomes in both cell types is likely similar. Although the putative relationship is limited by a paucity of comparative biochemical analyses of replication factors in different cell types, related insulator-driven control mechanisms for DNA replication may be conserved in endoreplicating and mitotically dividing diploid cells. The data thus implicates insulator/chromatin boundary elements as a critical attribute of DNA replication control. Our model suggests that delayed replication of repressed chromatin (e.g., intercalary heterochromatin) during very late S phase can be imposed in a simple, two-component mechanism. First, it requires that an extended genomic domain be completely devoid of functional origins of replication. The assembly and licensing of proximal pre-RC complexes can be repressed epigenetically or at the level of DNA sequence. Second, this domain is separated from flanking chromatin by a barrier element associated with an insulator complex, such as SUMM4. This structural organization is capable of preventing or delaying the entry of external forks fired from distal origins (Andreyeva, 2022).

An important frequent feature of the partially suppressed underreplication in mod(mdg4) animals is its asymmetry, which is consistent with a unidirectional penetration of the underreplicated domain by a replication fork firing from the nearest external origin. The SUMM4-dependent barrier may be created as a direct physical obstacle to MCM2-7 DNA-unwinding helicase or other enzymatic activities of the replisome. Alternatively, SUMM4 may inhibit the replication machinery indirectly by assembling at the insulator a DNA/chromatin structure that is incompatible with replisome translocation. This putative inhibitory structure may involve epigenetic modifications of chromatin as proposed earlier, linker histone H1 as shown previously and may also be dependent on Rif1, a negative DNA replication regulator that acts downstream of SUUR (Andreyeva, 2022).

In conclusion, this study used a newly developed MERCI approach to identify a stable stoichiometric complex termed SUMM4 that comprises SUUR, a previously known negative effector of replication, and Mod(Mdg4), an insulator protein. SUMM4 subunits cooperate to mediate transcriptional repression and chromatin boundary functions of gypsy-like (class 3) insulators and inhibit DNA replication likely by slowing down replication fork progression through the boundary element. Thus, SUMM4 is required for coordinate regulation of gene expression, chromatin partitioning, and DNA replication timing. The insulator-dependent regulation of DNA replication offers a novel mechanism for the establishment of replication timing in addition to the currently accepted paradigm of variable timing of replication origin firing (Andreyeva, 2022).

DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients (Kingsley, 2023).

Coordinating transcription and replication to mitigate their conflicts in early Drosophila embryos

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. It hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2 min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. It is proposed that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes (Cho, 2022).

Nucleoporins facilitate ORC loading onto chromatin

The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. This study perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, it was found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. This work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation (Richards, 2022).

list of proteins involved in DNA replication


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Marchal, C., Sima, J. and Gilbert, D. M. (2019). Control of DNA replication timing in the 3D genome. Nat Rev Mol Cell Biol 20(12): 721-737. PubMed ID: 31477886

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Das, S., Caballero, M., Kolesnikova, T., Zhimulev, I., Koren, A. and Nordman, J. (2021). Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila. Genetics 219(3). PubMed ID: 34740250

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Dewey, E. B., Korda Holsclaw, J., Saghaey, K., Wittmer, M. E. and Sekelsky, J. (2023). The effect of repeat length on Marcal1-dependent single-strand annealing in Drosophila. Genetics 223(1). PubMed ID: 36303322

Hiratani, I., et al. (2008). Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol. 6: e245. PubMed Citation: 18842067

Hou, C., Li, L., Qin, Z. S. and Corces, V. G. (2012). Gene density, transcription, and insulators contribute to the partition of the Drosophila genome into physical domains. Mol Cell 48(3): 471-484. PubMed ID: 23041285

Hua, B. L., Bell, G. W., Kashevsky, H., Von Stetina, J. R. and Orr-Weaver, T. L. (2018). Dynamic changes in ORC localization and replication fork progression during tissue differentiation. BMC Genomics 19(1): 623. PubMed ID: 30134926

Ji, J., Tang, X., Hu, W., Maggert, K. A. and Rong, Y. S. (2019). The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development. PLoS Genet 15(5): e1008169. PubMed ID: 31100062

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Kingsley, G., Skagia, A., Passaretti, P., Fernandez-Cuesta, C., Reynolds-Winczura, A., Koscielniak, K., Gambus, A. (2023). DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation. Nucleic Acids Res, 51(18):9748-9763 PubMed ID: 37638758

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date revised: 25 October 2017

Zygotically transcribed genes

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